As the methods for production of L-amino acids by direct fermentation, some methods are known for producing L-leucine by using microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium or Arthrobacter. Known methods for producing L-leucine by the use of microorganisms belonging to the genus Escherichia include: a method using a microorganism which is resistant to β-2-thienylalanine (Japanese Published Unexamined Patent Application No. 72695/81); a method using a microorganism which is resistant to L-ethionine (Japanese Published Unexamined Patent Application No. 55194/84); a method using a microorganism which is resistant to 2-ketobutyric acid (Japanese Published Unexamined Patent Application No. 9982/96); and a method using a microorganism which is resistant to 4-azaleucine or 5,5,5-trifluoroleucine (Japanese Published Unexamined Patent Application No. 70879/96).
Likewise, there are known methods for producing L-isoleucine by using microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium or Arthrobacter. Known methods for producing L-isoleucine by the use of microorganisms belonging to the genus Escherichia include: a method using a microorganism which is resistant to thiaisoleucine, isoleucine hydroxamate, arginine hydroxamate, DL-ethionine, etc. (Japanese Published Unexamined Patent Application No. 130882/93); a method using a microorganism which is resistant to 2-ketobutyric acid (Japanese Published Unexamined Patent Application No. 9982/96); and a method using a microorganism which grows rapidly in a medium containing L-homoserine as the only nitrogen source (Japanese Published Unexamined Patent Application No. 322583/96).
However, these methods have the problem that L-amino acids other than the desired L-amino acids are formed as by-products in considerable amounts. Particularly, formation of L-valine in the process of producing L-leucine or L-isoleucine causes the rise in production cost or the lowering of purification yield and product purity because separation and removal of L-valine in the purification step is not easy.
Alanine-valine transaminase (transaminase C) is an enzyme which catalyzes the reversible coupled transamination reaction between L-alanine and pyruvic acid, and 2-oxoisovaleric acid and L-valine, as shown below.L-alanine+2-oxoisovaleric acid⇄pyruvic acid+L-valine
This enzyme is also known to catalyze the reversible coupled transamination reaction between L-alanine and pyruvic acid, and 2-oxobutyric acid and 2-aminobutyric acid, as shown below.L-alanine+2-oxobutyric acid⇄pyruvic acid+2-aminobutyric acid
The gene encoding alanine-valine transaminase has been found in microorganisms such as Escherichia coli and Salmonella typhimurium. As for alanine-valine transaminase derived from Escherichia coli, there have been reports on the cloning of the gene encoding the enzyme (avtA) and the nucleotide sequence of the gene [J. Bacteriol., 169, 4228 (1987); Gene, 65, 195 (1988); Science, 277, 1453 (1997); Genbank, Accession No. AE00434 (1998)]. It is also reported that alanine-valine transaminase activity is enhanced by amplification of the avtA gene [J. Bacteriol. 169, 5610 (1987)].
The following have been reported on the physiological role of alanine-valine transaminase.
Escherichia coli which is deficient in ilvE encoding branched chain amino acid transaminase (transaminase B) exhibits the complete requirement for L-isoleucine, but not for L-valine (the leaky phenotype), indicating that alanine-valine transaminase is concerned in the conversion of 2-oxoisobutyric acid into L-valine as the second transaminase substituting for branched chain amino acid transaminase [Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington, D. C. (1987)]. Further, the partial requirement for L-valine (the leaky phenotype) of the above mutant is complemented by amplifying the avtA gene [Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington, D. C. (1987)].
However, there has been no report that the formation of L-valine can be reduced by enhancing the activity of alanine-valine transaminase.